CD4-Induced conformational changes in the human immunodeficiency virus type 1 gp120 glycoprotein: consequences for virus entry and neutralization

Human immunodeficiency virus type 1 (HIV-1) entry into target cells involves sequential binding of the gp120 exterior envelope glycoprotein to CD4 and to specific chemokine receptors. Soluble CD4 (sCD4) is thought to mimic membrane-anchored CD4, and its binding alters the conformation of the HIV-1 envelope glycoproteins. Two cross-competing monoclonal antibodies, 17b and CG10, that recognize CD4-inducible gp120 epitopes and that block gp120-chemokine receptor binding were used to investigate the nature and functional significance of gp120 conformational changes initiated by CD4 binding. Envelope glycoproteins derived from both T-cell line-adapted and primary HIV-1 isolates exhibited increased binding of the 17b antibody in the presence of sCD4. CD4-induced exposure of the 17b epitope on the oligomeric envelope glycoprotein complex occurred over a wide range of temperatures and involved movement of the gp120 V1/V2 variable loops. Amino acid changes that reduced the efficiency of 17b epitope exposure following CD4 binding invariably compromised the ability of the HIV-1 envelope glycoproteins to form syncytia or to support virus entry. Comparison of the CD4 dependence and neutralization efficiencies of the 17b and CG10 antibodies suggested that the epitopes for these antibodies are minimally accessible following attachment of gp120 to cell surface CD4. These results underscore the functional importance of these CD4-induced changes in gp120 conformation and illustrate viral strategies for sequestering chemokine receptor-binding regions from the humoral immune response.     

Oxidative stress and interleukins in seminal plasma during leukocytospermia

Various roles for the viral receptor, CD4, have been proposed in facilitating human immunodeficiency virus type 1 (HIV-1) entry, including virion binding to the target cell and the induction of conformational changes in the viral envelope glycoproteins required for the membrane fusion reaction. Here, we compare the structural requirements in the CDR2-like loop of CD4 domain 1, the major contact site of the gp120 envelope glycoprotein, for gp120 binding and virus entry. For every CD4 mutant examined, the level of cell surface expression and the gp120 binding affinity were sufficient to explain the relative ability to function as a viral receptor. The decrease in relative infectibility associated with decreased gp120 binding affinity was more pronounced at lower cell surface CD4 concentrations. These results imply that both receptor density and affinity determine the efficiency of HIV-1 entry and that specific structures in the CD4 residues examined are probably not required for HIV-1 entry functions other than gp120 binding.     

Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4(+) T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4(+) cells during gene therapy of AIDS.     

Spontaneous mutations in the env gene of the human immunodeficiency virus type 1 NDK isolate are associated with a CD4-independent entry phenotype

Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multistep process initiated by envelope protein gp120 binding to cell surface CD4. The conformational changes induced by this interaction likely favor a second-step interaction between gp120 and a coreceptor such as CXCR4 or CCR5. Here, we report a spontaneous and stable CD4-independent entry phenotype for the HIV-1 NDK isolate. This mutant strain, which emerged from a population of chronically infected CD4-positive CEM cells, can replicate in CD4-negative human cell lines. The presence of CXCR4 alone renders cells susceptible to infection by the mutant NDK, and infection can be blocked by the CXCR4 natural ligand SDF-1. Furthermore, we have correlated the CD4-independent phenotype with seven mutations in the C2 and C3 regions and the V3 loop. We propose that the mutant gp120 spontaneously acquires a conformation allowing it to interact directly with CXCR4. This virus provides us with a powerful tool to study directly gp120-CXCR4 interactions.     

Human immunodeficiency virus type 1 membrane fusion mediated by a laboratory-adapted strain and a primary isolate analyzed by resonance energy transfer

Previous studies of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein-mediated membrane fusion have focused on laboratory-adapted T-lymphotropic strains of the virus. The goal of this study was to characterize membrane fusion mediated by a primary HIV-1 isolate in comparison with a laboratory-adapted strain. To this end, a new fusion assay was developed on the basis of the principle of resonance energy transfer, using HeLa cells stably transfected with gp120/gp41 from the T-lymphotropic isolate HIV-1LA1 or the macrophage-tropic primary isolate HIV-1JR-FL. These cells fused with CD4+ target cell lines with a tropism mirroring that of infection by the two viruses. Of particular note, HeLa cells expressing HIV-1JR-FL gp120/gp41 fused only with PM1 cells, a clonal derivative of HUT 78, and not with other T-cell or macrophage cell lines. These results demonstrate that the envelope glycoproteins of these strains play a major role in mediating viral tropism. Despite significant differences exhibited by HIV-1JR-FL and HIV-1LAI in terms of tropism and sensitivity to neutralization by CD4-based proteins, the present study found that membrane fusion mediated by the envelope glycoproteins of these viruses had remarkably similar properties. In particular, the degree and kinetics of membrane fusion were similar, fusion occurred at neutral pH and was dependent on the presence of divalent cations. Inhibition of HIV-1JR-FL envelope glycoprotein-mediated membrane fusion by soluble CD4 and CD4-IgG2 occurred at concentrations similar to those required to neutralize this virus. Interestingly, higher concentrations of these agents were required to inhibit HIV-1LAI envelope glycoprotein-mediated membrane fusion, in contrast to the greater sensitivity of HIV-1LAI virions to neutralization by soluble CD4 and CD4-IgG2. This finding suggests that the mechanisms of fusion inhibition and neutralization of HIV-1 are distinct.     

Dissociation of the CD4 and CXCR4 binding properties of human immunodeficiency virus type 1 gp120 by deletion of the first putative alpha-helical conserved structure

To evaluate conserved structures of the surface gp120 subunit (SU) of the human immunodeficiency virus type 1 (HIV-1) envelope in gp120-cell interactions, we designed and produced an HIV-1 IIIB (HXB2R) gp120 carrying a deletion of amino acids E61 to S85. This sequence corresponds to a highly conserved predicted amphipathic alpha-helical structure located in the gp120 C1 region. The resultant soluble mutant with a deleted alpha helix 1 (gp120 DeltaalphaHX1) exhibited a strong interaction with CXCR4, although CD4 binding was undetectable. The former interaction was specific since it inhibited the binding of the anti-CXCR4 monoclonal antibody (12G5), as well as SDF1alpha, the natural ligand of CXCR4. Additionally, the mutant gp120 was able to bind to CXCR4(+)/CD4(-) cells but not to CXCR4(-)/CD4(-) cells. Although efficiently expressed on cell surface, HIV envelope harboring the deleted gp120 DeltaalphaHX1 associated with wild-type transmembrane gp41 was unable to induce cell-to-cell fusion with HeLa CD4(+) cells. Nevertheless, the soluble gp120 DeltaalphaHX1 efficiently inhibited a single round of HIV-1 LAI infection in HeLa P4 cells, with a 50% inhibitory concentration of 100 nM. Our data demonstrate that interaction with the CXCR4 coreceptor was maintained in a SUgp120 HIV envelope lacking alphaHX1. Moreover, in the absence of CD4 binding, the interaction of gp120 DeltaalphaHX1 with CXCR4 was sufficient to inhibit HIV-1 infection.     

Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies

Infection by some human immunodeficiency virus type 1 (HIV-1) isolates is enhanced by the binding of subneutralizing concentrations of soluble receptor, soluble CD4 (sCD4), or monoclonal antibodies directed against the viral envelope glycoproteins. In this work, we studied the abilities of different antibodies to mediate activation of the envelope glycoproteins of a primary HIV-1 isolate, YU2, and identified the regions of gp120 envelope glycoprotein contributing to activation. Binding of antibodies to a variety of epitopes on gp120, including the CD4 binding site, the third variable (V3) loop, and CD4-induced epitopes, enhanced the entry of viruses containing YU2 envelope glycoproteins. Fab fragments of antibodies directed against either the CD4 binding site or V3 loop also activated YU2 virus infection. The activation phenotype was conferred on the envelope glycoproteins of a laboratory-adapted HIV-1 isolate (HXBc2) by replacing the gp120 V3 loop or V1/V2 and V3 loops with those of the YU2 virus. Infection by the YU2 virus in the presence of activating antibodies remained inhibitable by macrophage inhibitory protein 1beta, indicating dependence on the CCR5 coreceptor on the target cells. Thus, antibody enhancement of YU2 entry involves neither Fc receptor binding nor envelope glycoprotein cross-linking, is determined by the same variable loops that dictate enhancement by sCD4, and probably proceeds by a process fundamentally similar to the receptor-activated virus entry pathway.     

The antigenic structure of the HIV gp120 envelope glycoprotein

The human immunodeficiency virus HIV-1 establishes persistent infections in humans which lead to acquired immunodeficiency syndrome (AIDS). The HIV-1 envelope glycoproteins, gp120 and gp41, are assembled into a trimeric complex that mediates virus entry into target cells. HIV-1 entry depends on the sequential interaction of the gp120 exterior envelope glycoprotein with the receptors on the cell, CD4 and members of the chemokine receptor family. The gp120 glycoprotein, which can be shed from the envelope complex, elicits both virus-neutralizing and non-neutralizing antibodies during natural infection. Antibodies that lack neutralizing activity are often directed against the gp120 regions that are occluded on the assembled trimer and which are exposed only upon shedding. Neutralizing antibodies, by contrast, must access the functional envelope glycoprotein complex and typically recognize conserved or variable epitopes near the receptor-binding regions. Here we describe the spatial organization of conserved neutralization epitopes on gp120, using epitope maps in conjunction with the X-ray crystal structure of a ternary complex that includes a gp120 core, CD4 and a neutralizing antibody. A large fraction of the predicted accessible surface of gp120 in the trimer is composed of variable, heavily glycosylated core and loop structures that surround the receptor-binding regions. Understanding the structural basis for the ability of HIV-1 to evade the humoral immune response should assist in the design of a vaccine.     

Expression of HIV env gene in a human T cell line for a rapid and quantifiable cell fusion assay

Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins present at the surface of infected cells are known to mediate fusion with CD4-positive target cells. In this study we have developed a novel Env-expressing cell line for investigating the fusion process in a biologically significant system. Cell surface expression of the HIV-1 env gene, isolated from the highly fusogenic strain SF33, was obtained in the CD4-negative T cell line A2.01. To render the system versatile and efficient, HIV-1 regulatory proteins Tat and Rev were supplied in trans. The presence of Env at the cell surface was shown by cytofluorometry and immunofluorescence and precursor processing of gp160 to gp120/gp41 was demonstrated by Western blot. The fusion capacity of A2.01-Env cells was assessed by coculture with CD4-positive T lymphocytes or the fusion indicator cell line, HeLa-CD4-LTR-beta-Gal. By coincubation with CD4-positive T cells such as SupT1, A2.01-Env cells were observed to mediate rapidly numerous well-defined syncytia in a reproducible fashion. By expressing Tat, they also had the capacity to trans-activate the LTR-linked reporter beta-Gal gene following fusion with HeLa-CD4-LTR-beta-Gal cells. The fusion-inhibiting anti-CD4 monoclonal antibodies Q425 and Q428 were used to block specifically Env-mediated fusion with CD4-positive cells and to demonstrate application of this system to the search for potential fusion-blocking agents. Our system thus offers a biologically significant model for studying fusion events with the advantages of being rapid, reproducible and versatile.     

Effect of mannosylated derivatives on HIV-1 infection of macrophages and lymphocytes

We previously demonstrated that gp120/160 (Env) of HIV-1 interact in a carbohydrate-specific manner with mannosyl/N-acetylglucosaminyl derivatives and that HIV-1LAI infection of monocytic U937 and lymphoid CEM cells was inhibited by CD4-free Concanavalin A-reactive glycopeptides from U937 cells. We report here that the natural glycoproteins bovine fetuin and asialofetuin, human orosomucoid and alpha-fetoprotein, and mannan, which all specifically interact with Env, inhibited infection of primary macrophages by macrophage-tropic HIV-1 strains, whereas dextran had no such effect. This activity was conserved if fetuin, asialofetuin, or orosomucoid were heat-treated, which rules out the role of their three-dimensional structure. Orosomucoid and mannan partially inhibited Env binding to macrophages but not to U937 or CEM cells. This indicates that Env does not bind in the same manner to primary macrophages and to immortalized CD4+ cells, and that orosomucoid and mannan act at CD4-independent stages of virus binding to macrophages. Mannan also inhibited Env binding to surface glycopeptides obtained after trypsin treatment of macrophages. Furthermore, orosomucoid and fetuin interacted with, and they inhibited the binding of a V3 loop B clade consensus peptide to several macrophage membrane proteins, including two 36 and 42 kDa proteins. These data indicate that these glycoproteins interfere with post-binding events during HIV-1 infection of primary macrophages. In contrast, the compounds did not affect infection of U937 or CEM cells by T-cell tropic HIV-1LAI nor infection of peripheral blood lymphocytes by HIV-1LAI or HIV-1(Ba-L). Thus, carbohydrate-specific inhibition of HIV infection depends on the cell type more than on the viral strain, and differences in the glycan structure of cell-type-specific cofactors may be important for HIV entry into cells.     

Identification of determinants on a dualtropic human immunodeficiency virus type 1 envelope glycoprotein that confer usage of CXCR4

The chemokine receptors CCR5 and CXCR4, in combination with CD4, mediate cellular entry of macrophage-tropic (M-tropic) and T-cell-tropic strains of human immunodeficiency virus type 1 (HIV-1), respectively, while dualtropic viruses can use either receptor. We have constructed a panel of chimeric viruses and envelope glycoproteins in which various domains of the dualtropic HIV-1(DH12) gp160 were introduced into the genetic background of an M-tropic HIV-1 isolate, HIV-1(AD8). These constructs were employed in cell fusion and virus infectivity assays using peripheral blood mononuclear cells, MT4 T cells, primary monocyte-derived macrophages, or HOS-CD4 cell lines, expressing various chemokine receptors, to assess the contributions of different gp120 subdomains in coreceptor usage and cellular tropism. As expected, the dualtropic HIV-1(DH12) gp120 utilized either CCR3, CCR5, or CXCR4, whereas HIV-1(AD8) gp120 was able to use only CCR3 or CCR5. We found that either the V1/V2 or the V3 region of HIV-1(DH12) gp120 individually conferred on HIV-1(AD8) the ability to use CXCR4, while the combination of both the V1/V2 and V3 regions increased the efficiency of CXCR4 use. In addition, while the V4 or the V5 region of HIV-1(DH12) gp120 failed to confer the capacity to utilize CXCR4 on HIV-1(AD8), these regions were required in conjunction with regions V1 to V3 of HIV-1(DH12) gp120 for efficient utilization of CXCR4. Comparison of virus infectivity analyses with various cell types and cell fusion assays revealed assay-dependent discrepancies and indicated that events occurring at the cell surface during infection are complex and cannot always be predicted by any one assay.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Infectious properties of human immunodeficiency virus type 1 mutants with distinct affinities for the CD4 receptor

Recent evidence suggests that primary patient isolates of T-cell-tropic human immunodeficiency virus type 1 (HIV-1 ) have lower affinities for CD4 than their laboratory-adapted derivatives, that this may partly result from tighter gp120-gp41 bonds that constrain the CD4 binding sites of the primary viruses, and that selection for increased CD4 affinity may be the principal factor in laboratory adaptation of HIV-1 (S. L. Kozak, E. J. Platt, N. Madani, F. E. Ferro, Jr., K. Peden, and D. Kabat, J. Virol. 71:873-882, 1997). These conclusions were based on studies with a panel of HeLa-CD4 cell clones that differ in CD4 levels over a broad range, with laboratory-adapted viruses infecting all clones with equal efficiencies and primary T-cell-tropic viruses infecting the clones in proportion to cellular CD4 levels. Additionally, all of the primary and laboratory-adapted T-cell-tropic viruses efficiently used CXCR-4 (fusin) as a coreceptor. To test these conclusions by an independent approach, we studied mutations in the laboratory-adapted virus LAV/IIIB that alter the CD)4 binding region of gp120 and specifically reduce CD4 affinities of free gp 120 by 85 to 98% (U. Olshevsky et al., J. Virol. 64:5701-5707, 1990). These mutations reduced virus titers to widely varying extents that ranged from severalfold to several orders of magnitude and converted infectivities on the HeLa-CD4 panel from CD4 independency to a high degree of CD4 dependency that resembled the behavior of primary patient viruses. The relative infectivities of the mutants correlated closely with their sensitivities to inactivation by soluble CD4 but did not correlate with the relative CD4 affinities of their free gp120s. Most of the mutations did not substantially alter envelope glycoprotein synthesis, processing, expression on cell surfaces, incorporation into virions, or rates of gp120 shedding from virions. However, one mutation (D457R) caused a decrease in gp160 processing by approximately 80%. The fact that several mutations increased rates of spontaneous viral inactivation (especially D368P) suggests that HIV-1 life spans may be determined by structural stabilities of viral envelope glycoproteins. All of the wild-type and mutant viruses were only slowly and inefficiently adsorbed onto cultured CD4-positive cells at 37 degrees C, and the gradual declines in viral titers in the media were caused almost exclusively by spontaneous inactivation rather than by adsorption. The extreme inefficiency with which infectious HIV-1 is able to infect cultured susceptible CD4-positive cells in standard assay conditions casts doubt on previous inferences that the vast majority of retrovirions produced in cultures are noninfectious. Apparent infectivity of T-cell-tropic HIV-1 in culture is limited by productive associations with CD4 and is influenced in an interdependent manner by CD4 affinities of viral gp120-gp41 complexes and quantities of cell surface CD4.     

Spontaneous and ligand-induced trafficking of CXC-chemokine receptor 4

A chimeric protein consisting of CXC-chemokine receptor 4 (CXCR4) and the green fluorescent protein (GFP) was used for studying receptor localization and trafficking in real time in stably transduced HeLa, U-937, CEM, and NIH/3T3 cells. CXCR4-GFP was fully active as a co-receptor in mediating human immunodeficiency virus (HIV) entry. Both CXCR4 and CXCR4-GFP were found to undergo significant spontaneous endocytosis. Only 51.5 +/- 7.8% of receptor molecules were found on the plasma membrane in CD4-positive cells, 43.9 +/- 8.5% were found in CD4-negative HeLa cells, 75.6 +/- 9.7% were found in U-937 cells, 72.5 +/- 7.9 were found in CEM cells, and almost none were found in in NIH/3T3 cells. Stromal cell-derived factor-1alpha induced rapid endocytosis of cell surface receptor molecules. A significant part of CXCR4 was targeted to lysosomes upon binding of the ligands, and recycling of internalized CXCR4 was not efficient. Only about 30% of receptor molecules recycled back to the cell surface in HeLa cells, 5% recycled in U937, and 10% recycled in CEM cells, suggesting that the protective effect of chemokines against HIV infection can be attributed not only to competition for binding but also to depletion of the co-receptor molecules from the cell surface. Envelope glycoprotein gp120 of syncytia-inducing/lymphocyte tropic HIV-1 strains induced rapid internalization of CXCR4 in both CD4-negative and CD4-positive cells, suggesting that gp120 is a high affinity ligand of CXCR4.     

Clustered localization of oligomeric Nef protein of human immunodeficiency virus type 1 on the cell surface

We studied human immunodeficiency virus type 1 (HIV-1) Nef protein biochemically and histologically. HIV-1 Nef, derived from baculosystem and from cells infected with HIV-1, formed homomeric monomers, dimers, trimers, and further polymers. These oligomers were non-covalently associated. In cells infected with HIV-1, Nef molecules were clustered at the cell surface as well as cytoplasm. Our previous results have indicated that the Nef on the surface of cells infected with HIV-1 is cytotoxic against uninfected CD4+ T cells. Thus, it is very likely that the HIV-1-mediated cytotoxic reaction is due, at least in part, to the clustered localization of oligomeric Nef on the cell surface.     

Epithelial uptake and transport of cell-free human immunodeficiency virus type 1 and gp120-coated microparticles

Cell-free human immunodeficiency virus type 1 (HIV-1) can be taken up and released by a monolayer of primary human gingival cells and remain infectious for CD4+ cells. Virus-sized latex particles covalently coated with purified native HIV-1 envelope glycoprotein gp120 are also transported through the primary epithelial cells. This process is significantly stimulated by increasing the intracellular cyclic AMP (cAMP) concentration. Inhibition experiments with mannan and alpha-methyl-mannopyranoside indicated that mannosyl groups are involved in the interaction between gp120 and gingival cells. An increase of cellular oligomannosyl receptors by incubation with the mannosidase inhibitor deoxymannojirimycin augmented transcellular transport of the gp120-coated particles. The results suggest that infectious HIV can penetrate gingival epithelia by a cAMP-dependent transport mechanism involving interaction of the lectin-like domain of gp120 and mannosyl residues on glycoproteins on the mucosal surface. Penetration of HIV could be inhibited by soluble glycoconjugates present in oral mucins.     

The role of complement and gp120-specific antibodies in virus lysis and CD4+ T cell depletion in HIV-1-infected patients

The substantial virus lysis was induced by HIV-1-infected patient serum and normal human complement serum in the presence of purified patient IgG. Non-infected CD4+ T cells coated with the whole virus or with a recombinant HIV-1 envelope gp120 and sensitised with patient IgG were also shown to be susceptible to complement-dependent lysis. The serum level of complement regulatory protein in a fluid phase, the C1-esterase inhibitor, was significantly correlated with serum concentration of C1q-circulating immune complexes (P=0.0062), but inversely with CD4+ T cell count (P < 0.0001). Accordingly, the disease progression in HIV-1-infected patients was significantly correlated with the level of complement activation as determined by serum level of C1-esterase inhibitor (P=0.0001), and inversely correlated with CD4+ cell count (P < 0. 0001) and gp120-specific antibody titre (P=0.0086). These results strongly suggest that the complement activation by gp120-specific antibodies play a very important role in virus clearance, but also in depletion of infected as well as gp120-coated non-infected CD4+ bystander T cells during the course of HIV-1 infection.     

Inhibition of CD4 translation mediated by human immunodeficiency virus type 1 envelope protein in a cell-free system

The human immunodeficiency virus type 1 (HIV-1) employs a number of complex strategies to interfere with the synthesis, stability, and subcellular localization of its specific cellular receptor CD4. To define better the mechanisms of inhibition of CD4 expression, we used a rabbit reticulocyte lysate in vitro system, in which cDNAs derived from HIV-1-infected cells were used to generate mRNA for the Tat, Vpu, and gp160 envelope proteins that were translated together with CD4-encoding mRNA. In the presence of microsomal membranes, we observed that cotranslation of Env mRNA resulted in a dose-dependent inhibition of CD4 translation. This effect was enhanced further when an mRNA-encoding Vpu in addition to Env mRNA was utilized. However, the activity of Vpu was mostly post-translational, since translation of Vpu alone, but not Env, was able to destabilize CD4 molecules presynthesized into microsomes. The Env-mediated inhibitory effect was specifically targeted at CD4 and did not affect the synthesis or stability of the CD8 molecule. Interestingly, mutated CD4 species, with a 20-fold lower affinity for HIV-1 Env than wild-type, were less sensitive to cotranslational inhibition. Our report identifies the envelope as the HIV-1 protein responsible for down-regulation of CD4 translation. We further propose a mechanism whereby direct interactions between gp160 and nascent CD4 molecules can cause interference with and premature termination of CD4 protein elongation.     

Monoclonal antibodies raised against covalently crosslinked complexes of human immunodeficiency virus type 1 gp120 and CD4 receptor identify a novel complex-dependent epitope on gp 120

The binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, gp120, to its cell surface receptor, CD4, represents a molecular interaction involving distinct alterations in protein structure. Consequently, the pattern of epitopes presented on the gp120-CD4 complex should differ from those on free gp120. To investigate this concept, mice were immunized with covalently crosslinked complexes of viral HIV-1IIIBgp120 and soluble CD4. Two monoclonal antibodies (MoAbs) obtained from the immunized mice exhibited a novel epitope specificity. The MoAbs were marginally reactive with HIV-1IIIBgp120, highly reactive with gp120-CD4 complexes, and unreactive with soluble CD4. The same pattern of reactivity was seen in solid-phase assays using HIV-1(451)gp120. A similar specificity for complexes was evident in flow cytometry experiments, in which MoAb reactivity was dependent upon the attachment of gp120 to CD4-positive cells. In addition, MoAb reactivity was detected upon the interaction of CD4 receptors with purified HIV-1IIIB virions. Notably, seroantibodies from HIV-positive individuals competed for MoAb binding, indicating that the epitope is immunogenic in humans. The results demonstrated that crosslinked gp120-CD4 complexes elicit antibodies to cryptic gp120 epitopes that are exposed during infection in response to receptor binding. These findings may have important implications for the consideration of HIV envelope-receptor complexes as targets for virus neutralization.     

Human studies in the development of human immunodeficiency virus vaccines

The safety and immunogenicity of candidate human immunodeficiency virus type 1 (HIV-1) vaccines have been studied in > 1500 healthy, seronegative (HIV-1-uninfected) subjects. HIV-1 envelope proteins, gp160 and gp120, have been the most extensively investigated. A live virus vector construct, vaccinia with insertion of the HIV-1 env gene, has also been studied. HIV-1 candidate vaccines have been well tolerated, with no acute or longer-term serious toxicity. Intramuscular multidose gp120 vaccines induce neutralizing antibodies, lymphoproliferative responses, and anti-HIV-1 CD4 cytotoxic T cell (CTL) activity. Immunization with the vaccinia-env construct, followed by a boost with an envelope protein, also induces neutralizing antibodies, and anti-HIV-1 CTL activity (CD8, major histocompatibility complex class I-restricted) has been observed. To date, serum from vaccinees can neutralize laboratory-adapted HIV-1 strains in vitro but not primary isolates; the significance of this observation is unknown. Additional approaches to vaccination against HIV-1 are in development.